MARINE ECOLOGY PROGRESS SERIES Vol. 103: 285-289,1994 Published January 10 Mar. Ecol. Prog. Ser.

Haemolymph nitrogen compounds and ammonia efflux rates under anoxia in the brackish water isopod Saduria entomon

Lars ~agerman',Anna szaniawska2

'Marine Biological Laboratory, Copenhagen University, Strandpromenaden 5. DK-3000 Helsinger, Denmark 20ceanographical Institute, Gdansk University, Al. Pilsudskiego 46, 81-378 Gdynia. Poland

ABSTRACT: Production and excretion of nitrogenous end products was studied in the benthic Baltic isopod Saduria entomon (L.) under normoxic and anoxic conditions. S. entomon is ammonotelic. Normoxic haemolymph ammonia concentrations were 176 +53 (SD) pm01 NH.,; I-' decreasing with time to 50 pm01 NH,' 1-' after 120 h anoxia. Normoxic ammonia efflux rate was 0.53 i- 0.07 (SD) pm01 NH,' g-' h-',decreasing 90% after 96 h anoxia. Normoxic haemolymph urate amounted to 135 pm01 I-', increasing after 120 h under anoxia to 741 +79 (SD) pm01 l-' Haemolymph urate concentrations were relatively high because of the ammonia excretion and an undetectable urate excretion even under anoxia. Circulating free amino acids also increased progressively under anoxia, from the nor- moxic 28 to 165 mm01 1'' after 96 h anoxia. This increase was largely due to accumulation of alanine as a result of an anaerobic metabolism. Ammonia was calculated to be the quantitatively dominat~ng nitrogenous end product in S. entomon. An effective efflux of ammonia prevents an excessive build-up of toxic ammonia in the haemolymph.

KEYWORDS: Ammonia . Amino acids . Urate . Total nitrogen . Blood . Excretion . Production

INTRODUCTION sent in amounts that normally permit the species to continue an aerobic metabolism (Hagerman & Vis- The brackish water isopod Saduria entomon has mann 1993). been the subject of several ecological and physio- In ammonotelic animals, ammonia dominates amongst logical studies made in recent years (e.g. Leonardsson the nitrogenous compounds which result from protein et al. 1987, Hagerman & Szaniawska 1988, 1990, 1991, metabolism and which are excreted under normoxia. 1992, Haahtela 1990, Vismann 1991). These studies Most crustaceans are ammonotelic and also excrete have revealed S. entomon to be a most adaptable spe- lesser amounts of amino acids, urea and uric acid cies with an exceptional tolerance of anoxia and (Regnault 1987). As long as the respiratory mechan- hydrogen sulphide concentrations. The extent of its isms are maintained, the normal nitrogen metabolism tolerance to periods of anoxia or to exposure of hydro- is also maintained. It is only when the overall meta- gen sulphide whilst under anoxia is dependent on its bolic rate is impaired that nitrogen metabolism may be activity pattern which determines whether the end expected also to change and when environmental con- product of the anaerobic metabolism will be lactate or ditions worsen to severe hypoxia or anoxia, a change alanine. Lactate accumulation relates to short-term in the production and efflux of nitrogenous waste survival whilst alanine accumulation leads to long- products may occur. It has been shown that increased term survival under anoxia (Hagerman & Szaniawska production of uric acid (urate) occurs in Carcinus 1990). In the presence of hydrogen sulphide, however, rnaenas and Callinectes sapidus when exposed to S. entomon invariably switches to anaerobiosis which hypoxia (Lallier et al. 1987, DeFur et al. 1990).Further- also leads to lactate production - even if oxygen is pre- more, it has been shown that anaerobic metabolism

O Inter-Research 1994 Mar. Ecol. Prog. Ser. 103: 285-289, 1994

can lead to increased haemolymph levels of amino Urate (uric acid) and urea: Haemolymph and water acids, with alanine being predominant, in Saduria urate and urea were measured using Sigma Diagnostic entornon (Hagerman & Szaniawska 1990). Kits 685 and 635 respectively. To correct for haemo- Although the respiratory responses of Saduria cyanin interference at the wavelength 520 nm a blank entomon have been studied intensively (Hagerman & absorbance measure was made on the same haemo- Szaniawska 1988, 1990, 1991, 1992), the origin and fate lymph volume as the test sample diluted in a buffer of the nitrogenous end products of this species are not identical to the one in the Sigma Kit. well known. The purpose of the present study was thus Amino acids: Amino acids in the haemolymph were to investigate ammonia efflux rates and haemolymph measured by HPLC (Jasco 880 PU) connected to a nitrogen compounds under anoxia in S. entomon. Perkin Elmer LC 1000 fluorescence detector. The ana- lytical method was pre-column (column: LiChrosorb RP18) derivatisation with orthophthalaldehyde (OPA) MATERIAL AND METHODS according to the description by Gardner & Miller (1980). Haemolymph (1 to 10 p1) was allowed to react Experimental procedure. Specimens of Saduria en- with 5 times as much OPA 2 min before injection. Indi- tomon (L.) were collected in the Gulf of Gdansk, Poland vidual amino acid calibration was made with a Sigma (S 7%) in 1991-92 and transported to the Marine Bio- amino acid calibration standard no. AA-S-18. logical Laboratory (Helsingnrr, Denmark) where they were stored at ambient temperature and salinity (T 7 "C; S 7 Yw).Specimens (except those starved for experimen- RESULTS tal purposes) were fed twice weekly with shrimp or bi- valve flesh. Anoxic conditions were maintained for up to Under normoxic conditions, the blood ammonia 120 h by bubbling the medium with N2 gas. Oxygen ten- concentrations in fed Saduria entomon are very variable sion of the medium was controlled by a microprocessor- but wlth a mean of 176 F 53 (SD) pm01 NH,' 1-' (Fig. 1). controlled regulator connected to a Radiometer PHM73 The exoskeleton of S. entomon is relatively heavier than acid-base analyser supplied with a Radiometer E5046 that of many other crustaceans and we may assume that oxygen electrode. The medium was considered to be the blood volume is correspondingly a smaller propor- anoxic when oxygen tensions were < 1 Torr. The aquaria tion of the fresh body weight. If 20 % of the wet weight is were covered with small plastic spheres. This dimin- taken to be a representative value for the blood volume ished contact with atmosphere and reduced the need for (Lockwood 1968, Spaargaren 1972), then the weight- N2 bubbling considerably. Change in water pH was thus specif~c blood concentration is 0.04 to 0.05 pm01 reduced to an increase of only 0.4 pH units (from pH 8.1 NH4+ g-' wet wt. Initially there was a difference be- to 8.5). tween the blood ammonia concentrations of the fed and In experiments involving the assessment of excre- starved groups with the latter having a mean of 100 to tion, Saduria entomon were kept individually in con- 120 pm01 NH4+1-' (0.02 to 0.03 pm01 NH,' g-' wet wt). tainers with 250 m1 of the medium. These solutions These lower values in the starved specimens are pos- were kept anoxic by bubbling N2 gas through them as sibly due to a decline in protein catabolism reducing described above. Water samples were taken at timed intervals and either analysed immediately or kept frozen at -80°C untll needed for analysis. Haemolymph samples were collected by inserting a hypodermic syringe (Terumo, 100 p1) into the heart region from d dorsoposterior direction. Each individual was sampled once only. Analytical procedure. Total ammonia (NH,'): Ammonia concentrations were measured using a modi- fied flow injectiodgas diffusion technique (Clinch et al. 1988) as described by Hosie et al. (1991) and Hunter & Uglow (1993a). Water samples were injected directly into the NaOH carrier stream and blood samples (25 ,u1) 0 1 10 were diluted 1 :40 before injection. The peak heights of 0 24 48 72 96 120 the chart outputs of the water and blood samples were T~mein anoxia (hours) compared with those of a series of fresh ammonium suI- Fig. 1 Saduna entomon. Haemolymph ammonia concen- phate standards in the range 5 to 25 pm01 NH,+l-l pre- trations in normoxia and as a function of t~meunder anoxia. pared daily with the medium or saline respectively. (m)pm01 l ' haemolymph; (0) pm01 g.' wet wt Hagerman & Szaniawska. Ammonia efflux by Saduna entomon under anoxia

ammonia production or to starvation-induced tissue Table 1. Sadul-ia entomon. Haemolymph ammonia production shrinkage causing an increased blood volume. Blood and haemolymph ammonla replacement times ammonia levels decrease with time under anoxia and in fed isopods drop quickly over the first 24 h to values sim- Product~on Replacement time ilar to those of the starved specimens. In both fed and (pm01 g-' wet wt h-') (mln) starved groups, the mean blood ammonia values after Normoxia 0.54 120 h under anoxia had dropped to a mean of 50 pm01 24 h anoxia 0.48 NH,' 1-' (0.011 pm01 NH,+ g-' wet wt) - a >4-fold 48 h anoxia 0.37 change from normoxic values. 72 h anoxia 0.18 The mean normoxic weight-specific ammonia efflux 96 h anoxia 0.1 rate was 0.53 ? 0.07 (SD) pm01 NH,' g-' h-' (Fig. 2). Efflux rates showed a wide variability and no differ- ence between the means of the starved and fed groups No urea was detected in any of the water or blood was found. The efflux rates decreased steadily after samples collected under normoxia or whilst under 24 h under anoxia and after 96 h reached 0.052 +0.017 anoxia. Small quantities of urate were present in the (SD) pm01 ammonia g-' h-'. This represents a 90% haemolymph samples collected under normoxia and drop from original rates, corresponding to a decrease these amounted to 135 ? 21 (SD) ~molurate 1-' haemo- of 4.7 nmol h-' or 0.9 % h-' of the normoxic efflux rate. lymph (Fig. 3).The urate concentrations increased pro- The weight-specific blood ammonia concentration gressively during the anoxic period and reached 74 1 i and excretion rate can be used to derive a turnover 79 pm01 urate 1-' haemolymph (= 0.15 pm01 urate g-' ratio - the time required to effect a complete replace- wet wt) after 120 h. Excretion of urate was not noticed; ment of the blood ammonia. Under steady state, urate water concentration was below the detection weight-specific blood ammonia production equals limit. excretion. Under normoxia, the ammonia replacement The total concentration of circulating free amino time for Saduna entomon under the same salinity/ acids under normoxia was 28.1 mm01 1-' haemolymph, temperature conditions used here is 6.9 mm. This rep- increasing steadily under anoxia to reach 165.1 mm01 resents a tissue ammonia production of 0.55 pm01 NH,' 1-' after 96 h. This large increase was due mainly to g-' wet wt h-'. After 96 h under anoxia, the replace- an accumulation of alanine which, as a result of an- ment rate became 20.7 min which equates to a tissue aerobic metabolism, increased from the normoxic production rate of 0.1 pnlol NH:' g-' wet wt h-'. Data 4.7 to 100 mm01 1-' by the end of the anoxic period. obtained on ammonia production and replacement Data relating to the haemolymph concentrations of rates during the experiments are given in Table 1. the measured variables are given in Table 2 which These data show a lower or no initial decrease in pro- shows the anoxia-induced accumulation of urate and duction (= excretion) rates which probably reflect the amino acids. This resulted in an increased ratio of the stress-induced elevation of metabolic rates which urate-N:ammonia-N in the haemolymph over the accompanied the transfer of the isopods to the experi- anoxic period, urate-N is 57 times higher than am- mental conditions. monia-N after 120 h anoxia. This is likely caused

Z 0 I I I l I 0 24 48 72 96 0 24 48 72 96 120 T~meIn anoxra (hours) T~meIn anoxla (hours)

Fig 2. Saduna entomon. Ammonia excretion (pmol g-' wet kvt Fig 3. Saduria entomon. Haemolymph urate concentra- h-'; mean f SD) under nor~noxiaand as a function of time tions in normoxla and as a function of time under anoxia. under anoxla (m) pm01 l-' haemolymph; (m)pm01 g-' wet wt 288 Mar. Ecol. Prog. Ser. 103: 285-289, 1994

were calculated. This time is short com- Table 2. Sadunaentomon. Haernolymph concentrations (lmol I-])of nitro- pared with that for Nephrops norvegicus genous compounds in normoxia and after 120 h anoxia (96 h for FAA) (Hosie et al. 1991) but similar to that of Crangon crangon (Hunter & Uglow Urate Urea Amino acids Alanine 1993b) which probably reflects the activ- (% FAA) ity/metabolic level of the species. The Normoxia 176 +53 135 f 21 28 100 combination of the lowered haemolymph Anoxia 120 h 50 f 5 74 1 +79 165 100 60.5 ammonia levels and the decreased efflux rate found after 96 h anoxia indicates ammonia production inhibition and paral- entirely by an efficient ammonia excretion and a lels the decrease in metabolic levels found in S. negligible urate excretion. Assuming no normoxic entomon under such anoxia (Hagerman & Szaniawska ammonia excretion (normally ca 0.5 pm01 NH4+g-' h-') 1990). during a 24 h period and an initial haemolymph con- According to Regnault (1987) some 1 to 5 % of the centration of 0.04 pm01 NH4+ g-', the accumulated end products of nitrogen metabolism could be urea. blood ammonia would be 12.4 pm01 NH,+ g-' after Urea was, however, not detected in the haemolymph of 24 h. This should be compared to an accumulated Saduria entomon, either under normoxia or anoxia but urate of ca 0.02 pm01 g-' or a production of 0.1 nmol a conspicuous production of urate was found under g-' h-', a value practically negligible compared to the anoxia. The final urate concentrations in the haemo- ammonia production of up to 0.54 pm01 g-l h-'. When lymph were high relative to those of blood ammonia using anoxic excretion and blood ammonia values, the (Table 2), mainly because of the efficiency of the corresponding accumulated blood ammonia would be ammonia excretion even under anoxia. 3.6 pm01 g-l, still accentuating the importance of the Lallier et al. (1987) found a significant increase in ammonia excretion in preventing excessive build-up of Carcinus maenas haemolymph urate after 24 h under nitrogenous by-products in the haemolymph. hypoxia (20 Torr). Urate has been found also In other species when subjected to hypoxia, e.g. Penaeus japonicus (Lallier & Truchot 1989), Homarus vulgaris DISCUSSION (Nies et al. 1992) and Nephrops norvegicus (H.Schliit- ter & L. Hagerman unpubl.). It may thus be that anaer- The ammonia efflux rate in crustaceans is size- obiosis-induced haemolymph urate accumulation is a dependent (Regnault 1987) and the normoxic weight widespread phenomenon in benthic crustaceans. specific ammonia efflux rates found here for Saduria The normoxic levels of haernolymph free amino entomon (mean = 0.5 lmol NH,' g-' h-') are in the acids (FAA) found in Saduria entomon lie within the normal range found for crustaceans (0.2 to 1.6 pm01 range found for higher crustaceans. Free-living marine g-' h-'; Regnault 1986). The individual variation of (brackish water) isopods seem to have haemolymph efflux rates probably reflects individual differences in FAA concentrations higher than those of most deca- metabolic rate due to activity and nutntional differ- pods (Charmantier et al. 1976) and th~salso appears to ences. A reduced rate of efflux under severe hypoxia be the case for S. entomon. The normoxic amino acids has been observed to occur in some species and (especially glycine, proline and alanine) help to main- may be a general trend amongst crustaceans (Reg- tain the cellular osmotic pressure (Weber & van Mar- nault & Aldrich 1988). The decrease in efflux rate rewijk 1972) and haemolymph amino acids are also the found to occur here during anoxla seems to follow this products of protein turnover (Claybrook 1983). The trend. anoxia-induced increase in haemolymph FAA is prob- The normoxic haernolymph ammonia concentrations ably due to protein catabolism as anoxia is invariably found here for Saduria entomon ranged from 115 to associated with starvation and an accumulation of ala- 250 pm01 NH; 1" and also compare well with what is nine as an anaerobic end product (Hagerman & Szani- known for other (mainly decapod) crustaceans (Hager- awska 1990). Although haemocyanin is also broken man et al. 1990). The concentrations are however down under anoxia it is unlikely to be a major contrib- much lower when expressed as weight specific (mean utor to the FAA build-up because it is not particularly 0.04 pm01 ammonia g-' wet wt). Assuming that the rich in proline, glycine and alanine (Boone & Schoffe- blood volume of S. entomon is approx~mately 20% niels 1979). (which is slightly less than values quoted for decapod The present study confirms that Saduria entomon species, e.g. Spaargaren 1972, because of the rela- can tolerate a considerable time under anoxia and this tively greater exoskeleton weight in these isopods), presumably has considerable survival value to a mud- then normoxic ammonia replacement times of 6.9 min dwelling organism. During anoxia there appears to be Hagerman & Szan~awska:Ammonia efflux by Saduria entomon under anoxia

an enhanced build-up of urate and certain free amino Hagerman, L.. Szaniawska. A. (1992). Saduria entomon: eco- acids in the haemolymph and a reduced but still effec- physiological adaptations for survival in the Baltic. In: tive efflux of ammonia. Presumably, this combination Bjarnestad. E.. Hagerman, L.,Jensen, K (eds.) Proc. 12th Baltic Marine Biologists Symp Olsen & Olsen, Fredens- of strategies effects a removal of nitrogenous waste borg, p 71-76 and minimises the attendant problems of an excessive Hagerman, L., Vlsmann, B (1993). Anaerobic metabolism, build-up of potentially toxic ammonia in the haemo- hypoxla and hydrogen sulph~de In the brackish water lymph. isopod Sadul-)a entomon (L). Ophelia 38 1-11 Hos~e,D. A., Uglow, R. F, Hagerman, L., Sondergaard, T., Welle, K (1991). Some effects of hypox~aand medium am- Acknowledgement. The study was supported by the Danish monia enrichment on efflux rates and circulating levels of Sclence Research Council grant no. 11.-8391. ammonia in Nephrops non/egicus. Mar. Blol. 110: 273-279 Hunter, D., Uglow, R. F. (1993a) A technique for the measurement of total ammonia in small volunles of sea- LITERATURE ClTED water and haemolymph. Ophelia 37 31-40 Hunter, D., Uglow, R F (1993b). Moult stage-dependent Boone, W. R., Schoffeniels, E. (1979). Hemocyanin synthesis variability of haemolymph ammonia and total protein during hypo-osmotic stress in the shore crab Carcjnus levels of Crangon crangon. Ophel~a37. 41-50 nlaenas (L.).Comp. Biochem. Physiol. 63B: 207-214 Lallier, F., Boitel, F., Truchot, J P. (1987). The effect of Charmantier, G., Charmantier, M., Voss-Foucart, M.-F., ambient oxygen and temperature on haemolymph Jeuniaux, C. (1976). Les acides amines libres de l'hemo- l-lactate and urate concentrations in the shore crab lymphe des isopodes marins Sphaeroma hookeri, Sphae- Carcinusmaenas. Comp. Biochem. Physiol. 86A: 255- 260 roma serratum (Flabellifera) et Idotea baltica (Valvifera). Lallier, F., Truchot, J. P. (1989). Modulation of haemocyanin Arch. int. Physiol. Biochim. 84: 989-996 oxygen-affinity by l-lactate and urate in the prawn Claybrook, D. L. (1983). Nitrogen metabolism. In: Mantel, L. Penaeus japonicus. J. exp. Biol. 147: 133-146 (ed.) The biology of Crustacea, Vol. 5, Internal anatomy Leonardsson, K., Bengtsson, A.,Linner, J. (1987). Size selec- and physiological regulation. Academic Press, New York, tive predation by fourhorn sculpin, Mjfoxocephalus p. 163-213 quadricornis (L.) (Pisces) on Mesidotea entomon (L.) Clinch, J. R., Worsfold, P. J., Sweetinq, F. W. 11988). An auto- (Crustacea, Isopoda). Hydrobiologia 164: 213-220 mated spectrophotometric field monitor fbr water quality Lockwood. A. P. M. (1968). Aspects of the physiology of parameters. Determination of ammonia. Analvt. chim. Crustacea. Oliver & Boyd. Edinburgh Acta 214: 401-407 Nies. A., Zeis. B.. Bridges. C. R.. Grieshaber, M. K. (1992). DeFur, P. L., Mangum, C. P., Reese, J. E. (1990). Respiratory Allosteric modulation of haemocyanin oxygen affinity by responses of the blue crab Callinectes sapidus to long- I-lactate and urate in the lobster Homarus vulgaris. 11. term hypoxia. Biol. Bull. 178: 46-54 Characterisation of specific effector binding sites. J. exp. Gardner, W. S., Miller, W. H. (1980). Reverse-phase liquid Biol. 168: 111-124 chromatographic analysis of amino acids after reaction Regnault, M. (1986). Production d'NH,+ par la crevette Cran- with o-phthalaldehyde. Analyt. Biochem. 101: 61-65 gon crangon L. dans deux ecosystemes c6tiers. Approche Haahtela, I. (1990). What do Baltic studies tell us about the experimentale et etude de l'influence du sediment sur le isopod Saduria entomon? 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(1991).The physiology of sulfide detoxification in strategy of the glacial relict isopod Saduria (Mesidotea) the isopod Saduria (Mesjdotea) entomon (L.). Mar. Ecol. entornon. Mar. Ecol. Prog. Ser. 59: 91-96 Prog. Ser. 76: 283-292 Hagerman, L., Szaniawska, A. (1991). lon regulation under Weber, R. E., van Marrewijk, W. J. A. (1972). Free amino acids anoxia in the brackish water isopod Saduria (Mesidotea) in the shrimp Crangon crangon and their osmoregulatory entomon. Ophelia 33: 97-104 significance. Neth. J. Sea Res 5: 391-415

This article was submitted to the editor Manuscript first received: A4arch 22, 1993 Revised rferslon accepted: October 13, 1993